ABSTRACT In the human genome, alternative splicing events facilitate the generation of a proteome with a greater diversity than is observed in the protein-coding gene repertoire. In other words, the vast majority of human genes can each produce numerous different transcripts and subsequently multiple protein isoforms. However, aberrant splicing can produce an increase in novel isoforms or normally low-level isoforms leading to potentially detrimental effects including altered protein function, protein- protein interactions as well as remodeling of protein complexes and pathways. In fact, the increase in these alternatively-spliced isoforms is now recognized as a major contributor to oncogenic phenotypes, such as the development of tumors and new blood vessels by supporting cell invasion and proliferation. A central challenge to realizing the biological impact, prognostic and therapeutic potential of alternatively-spliced isoforms across multiple cancer types has been a lack of technology available for unambiguously differentiating highly similar protein variants in their native forms. Current methods of detecting and studying alternatively-spliced isoforms at the protein level rely solely on indirect methods such as tag-based detection and nonspecific affinity reagents that are unable to discriminate among multiple protein isoforms. Herein, we propose a novel approach to develop highly specific antibodies against alternatively-spliced protein isoforms using a targeting method at the splice site junction with near amino acid specificity. With this technology, antibodies can be generated to specifically detect a desired, alternatively-spliced protein isoform without cross-reactivity to the native full-length form. This technological advance will accelerate oncology research and enable scientists to study both high-value and novel protein isoforms and their direct role in specific cancer phenotypes, metastatic potential, tumor grade specificity and survival rates, and provide a validation means for assessing the therapeutic potential of anticancer alternative-splicing inhibitors.